Bacterial 16S rRNA gene sequencing studies are popular across many fields of biology. This technique has allowed us to study bacterial communities like never before, leading to significant insights into microbial ecology and host- microbe interactions. However, 16SrRNAgene-based workflows are vulnerable to confounding and bias at every step. Many studies are plagued by entrenched methodological errors, producing data riddled with experimental artefacts. These issues are amplified in the study of low bacterial biomass samples, such as forensic and ancient samples, blood, meconium, ice and the built environment. It is, therefore, necessary to define the pitfalls of low biomass 16S rRNA gene-based work flows and to identify methods that may allow more accurate characterisation of bacterial communities in such samples.